Compositions and methods to treat Niemann-Pick disease

ABSTRACT

The present disclosure is directed to compositions comprising bryostatin-1, and methods comprising administering a composition comprising bryostatin-1, to treat Niemann-Pick Type C in a subject in need thereof.

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/971,480, filed Mar. 27, 2014, the contents of which areincorporated herein by reference.

Niemann-Pick Type C (NPC) is an inherited metabolic disorder known as alipid storage disease. It is caused by mutations in the NPC1 or NPC2gene. The proteins produced from these genes are involved in themovement of lipids, such as cholesterol, between cells. The genemutations disrupt the transport of cholesterol and other lipids betweencells resulting in excessive accumulation of lipids within tissues andorgans, eventually leading to cell death and the malfunction of majororgan systems, including the brain. The progressive deterioration of thenervous system is ultimately fatal. NPC may appear early in life ordevelop in the teen or adult years. Affected individuals have moderateenlargement of the spleen and liver, and brain damage may be extensiveand cause an inability to look up and down, difficulty in walking andswallowing, and progressive loss of vision and hearing. NINDSNiemann-Pick Disease Information Page, available athttp://www.ninds.nih.gov/disorders/niemann/niemann.htm; Niemann-Pickdisease, available athttp://ghr.nlm.nih.gov/condition/niemann-pick-disease.

There is currently no cure for NPC. Thus, there is a need to developtherapeutic agents to treat and/or minimize its associated symptoms.Further, it has been reported that the intermediate filament vimentin ishypophosphorylated in NPC-diseased cells compared to Wt cells and thatthis hypophosphorylation results from reduced protein kinase C (PKC)activity, in particular the α, ε, and βII isoforms. Tamari et al., PKCActivation in Niemann Pick C1 Cells Restores Subcellular CholesterolTransport, PLOS ONE, Vol. 8, Iss. 8 (2013). As Tamari et al. explain,cells lacking vimentin are unable to transport LDL-derived cholesterolfrom their lysosomes to the endoplasmic reticulum for esterification,and decreased vimentin phosphorylation reduces the pool of solublevimentin. Id. Increased PKC expression, in particular, the α, ε, and/orβII isozymes, phosphorylates vimentin and increases levels of solublevimentin in NPC-diseased cells, ameliorating the cholesterol transportblock. Id. As shown in FIGS. 7 and 8, PKC ε and α have beensubstantially detected in the brain (Wetsel et al., Journal of CellBiology, Vol. 117, 1992), which suffers progressively significantdegeneration in NPC. Thus, there is a further need to discover anddevelop agents that act as potent activators of particular PKC isoforms,such as PKC ε and/or α, to treat Niemann-Pick disease.

Accordingly, the present disclosure is directed to a compositioncomprising bryostatin-1 in an effective amount to treat NPC, wherein theeffective amount results in a concentration of bryostatin-1 in the brainof a subject in need of treatment ranging from about 0.1 nM to about 1.5nM.

The present disclosure is also directed to a method for treating NPC ina subject in need of treatment, comprising administering to the subjecta composition comprising bryostatin-1 in an amount effective to treatNPC, wherein the effective amount results in a concentration ofbryostatin-1 in the brain ranging from about 0.1 nM to about 1.5 nM.

Additional advantages of the disclosure will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the disclosure will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe disclosure and together with the description, serve to explain theprinciples of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dose kinetics and PKC isozyme specificity of bryostatin-1in primary human neurons.

FIGS. 2A and 2B show time kinetics and PKC isozyme specificity of 0.25nM bryostatin-1 in primary human neurons.

FIG. 3 shows PKC isozyme specificity of 0.27 nM Bryostatin-1 in SH-SY5Ycells.

FIGS. 4A-4C show PKC isozyme activation by other PKC activators(DHA-CP6-ME (4A), DCP-LA (4B) and DCPLA-ME (4C)) at variousconcentrations.

FIGS. 5A and 5B show PKC-ε and PKC-α activation by bryostatin-1 in mousebrain at various doses after 30 minutes or 120 minutes.

FIG. 6A and 6B show the time course of PKC-ε and PKC-α activation,respectively, by bryostatin-1 in mouse brain at certain doses.

FIG. 7 shows the distribution of PKC α and PKC ε in the body, asprovided in Wetsel et al., Journal of Cell Biology, Vol. 117 (1992).

FIG. 8 shows the relative abundance of PKC isozymes in rat tissue, asprovided in Wetsel et al., Journal of Cell Biology, Vol. 117 (1992).

DESCRIPTION

Particular aspects of the disclosure are described in greater detailbelow. The terms and definitions as used in the present application andas clarified herein are intended to represent the meaning within thepresent disclosure. The patent and scientific literature referred toherein is hereby incorporated by reference. The terms and definitionsprovided herein control, if in conflict with terms and/or definitionsincorporated by reference.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context dictates otherwise.

The terms “approximately” and “about” mean to be nearly the same as areferenced number or value including an acceptable degree of error forthe quantity measured given the nature or precision of the measurements.As used herein, the terms “approximately” and “about” should begenerally understood to encompass ±20% of a specified amount, frequencyor value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that term “about” or “approximately” can beinferred when not expressly stated.

The terms “administer,” “administration,” or “administering” as usedherein refer to (1) providing, giving, dosing and/or prescribing byeither a health practitioner or his authorized agent or under hisdirection a composition according to the disclosure, and/or (2) puttinginto, taking or consuming by the patient or person himself or herself, acomposition according to the disclosure. As used herein,“administration” of a composition includes any route of administration,including oral, intravenous, subcutaneous, intraperitoneal, andintramuscular.

As used herein, the term “subject” means a mammal, i.e., a human or anon-human mammal.

As used herein, “protein kinase C activator” or “PKC activator” means asubstance that increases the rate of the reaction catalyzed by proteinkinase C by binding to the protein kinase C.

The term “pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a subject.

Protein kinase C (PKC) is one of the largest gene families ofnon-receptor serine-threonine protein kinases. Since the discovery ofPKC in the early eighties and its identification as a major receptor forphorbol esters, a multitude of physiological signaling mechanisms havebeen ascribed to this enzyme. Kikkawa et al., J. Biol. Chem. (1982),vol. 257, pp. 13341-13348; Ashendel et al., Cancer Res. (1983), vol. 43:4333-4337. The interest in PKC stems from its unique ability to beactivated in vitro by calcium and diacylglycerol (and phorbol estermimetics), an effector whose formation is coupled to phospholipidturnover by the action of growth and differentiation factors. Activationof PKC involves binding of 1,2-diacylglycerol (DAG) and/or1,2-diacyl-sn-glycero-3-phospho-L-serine (phosphatidyl-L-serine, PS) atdifferent binding sites. An alternative approach to activating PKCdirectly is through indirect PKC activation, e.g., by activatingphospholipases such as phospholipase Cγ, by stimulating the Ser/Thrkinase Akt by way of phosphatidylinositol 3-kinase (PI3K), or byincreasing the levels of DAG, the endogenous activator. Nelson et al.,Trends in Biochem. Sci. (2009) vol. 34, pp. 136-145. Diacylglycerolkinase inhibitors, for example, may enhance the levels of the endogenousligand diacylglycerol, thereby producing activation of PKC. Meinhardt etal., Anti-Cancer Drugs (2002), vol. 13, pp. 725-733. Phorbol esters,however, are not suitable compounds for eventual drug developmentbecause of their tumor promotion activity. Ibarreta et al. Neuroreport(1999), vol. 10, pp. 1035-1040).

The PKC gene family consists of 11 genes, which are divided into foursubgroups: (1) classical PKC α, β1, β2 (β1 and β2 are alternativelyspliced forms of the same gene) and γ; (2) novel PKC δ, ε, η, and θ; (3)atypical PKC ζ and ι/λ; and (4) PKC_(μ). PKC_(μ) resembles the novel PKCisoforms but differs by having a putative transmembrane domain. Blobe etal. Cancer Metastasis Rev. (1994), vol. 13, pp. 411-431; Hug et al.Biochem. J. (1993) vol. 291, pp. 329-343; Kikkawa et al. Ann. Rev.Biochem. (1989), vol. 58, pp. 31-44. The classical PKC isoforms α, β1,β2, and γ are Ca²⁺, phospholipid, and diacylglycerol-dependent, whereasthe other isoforms are activated by phospholipid, diacylglycerol but arenot dependent on Ca²⁺ and no activator may be necessary. All isoformsencompass 5 variable (VI-V5) regions, and the α, β, and γ isoformscontain four (C1-C4) structural domains which are highly conserved. Allisoforms except PKC α, β, and γ lack the C2 domain, the ι/λ and ηisoforms also lack nine of two cysteine-rich zinc finger domains in C1to which diacylglycerol binds. The C1 domain also contains thepseudosubstrate sequence which is highly conserved among all isoforms,and which serves an autoregulatory function by blocking thesubstrate-binding site to produce an inactive conformation of theenzyme. House et al., Science (1987), vol. 238, pp. 1726-1728.

Because of these structural features, diverse PKC isoforms are thoughtto have highly specialized roles in signal transduction in response tophysiological stimuli as well as in neoplastic transformation anddifferentiation. Nishizuka, Cancer (1989), vol. 10, pp. 1892-1903;Glazer, pp. 171-198 in Protein Kinase C, I. F. Kuo, ed., Oxford U.Press, 1994. For a discussion of PKC modulators see, for example,International Application No. PCT/US97/08141 (WO 97/43268) and U.S. Pat.Nos. 5,652,232; 6,080,784; 5,891,906; 5,962,498; 5,955,501; 5,891,870and 5,962,504, each incorporated by reference herein in its entirety.

PKC activators can be broad-spectrum activators, acting on multipleisoforms of PKC, or can be selective for certain isoforms. Selective PKCactivators may offer unique advantages because different isoforms canperform different functions. It has been shown that increased PKCexpression of the α, ε, and/or βII isoforms phosphorylates vimentin andincreases levels of soluble vimentin in Niemann-Pick diseased cells,ameliorating the cholesterol transport block associated with thedisease. Tamari et al., PKC Activation in Niemann Pick C1 Cells RestoresSubcellular Cholesterol Transport, PLOS ONE, Vol. 8, Iss. 8 (2013). Thepresent inventor has discovered that a concentration of bryostatin-1 inthe brain within the range of 0.1 nM to 1.5 nM results in a PKC isozymeactivation signature (i.e., the activation of specific PKC isozymes,including PKC ε and/or α) that is particularly advantageous for treatingNPC.

Thus, in one aspect of the present disclosure, a composition comprisesbryostatin-1 in an effective amount to treat NPC, wherein the effectiveamount results in a concentration of bryostatin-1 in the brain of asubject in need of treatment ranging from about 0.1 nM to about 1.5 nM.For example, in some embodiments, the effective amount results in aconcentration of bryostatin-1 in the brain of about 0.1 nM, about 0.2nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM,about 1.3 nM, about 1.4 nM, about 1.5 nM, or any value in between.

The blood plasma concentration of bryostatin-1 corresponds to about fivetimes the concentration of bryostatin-1 in the brain. Thus, in someembodiments, an effective amount of bryostatin-1 to treat NPC results ina blood plasma concentration of about 0.5 nM to about 7.5 nM. Forexample, in some embodiments, the effective amount of bryostatin-1results in a blood plasma concentration of bryostatin-1 of about 0.5 nM,about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about3.5 nM, about 4 nM, about 4.5 nM, about 5 nM, about 5.5 nM, about 6 nM,about 6.5 nM, about 7 nM, about 7.5 nM, or any value in between.

In some embodiments, the effective amount of bryostatin-1 to treat NPCranges from about 5 μg/m² to about 120 μg/m². For example, in someembodiments, the effective amount of bryostatin-1 to treat NPC is about5 μg/m², about 10 μg/m², about 15 μg/m², about 20 μg/m², about 25 μg/m²,about 30 μg/m², about 35 μg/m², about 40 μg/m², about 45 μg/m², about 50μg/m², about 55 μg/m², about 60 μg/m², about 65 μg/m², about 70 μg/m²,about 75 μg/m², about 80 μg/m², about 85 μg/m², about 90 μg/m², about 95μg/m², about 100 μg/m², about 105 μg/m², about 110 μg/m², about 115μg/m², about 120 μg/m², or any value in between.

In some embodiments, the concentrations of bryostatin-1 disclosed hereinare peak concentrations of bryostatin-1.

Formulations of the pharmaceutical compositions described herein may beprepared by any suitable method known in the art of pharmacology. Ingeneral, such preparatory methods include bringing the activeingredient, i.e., bryostatin-1, into association with a carrier or oneor more other accessory ingredients, then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions suitable forethical administration to humans, it will be understood by skilledartisan that such compositions are generally suitable for administrationto animals of all sorts. Modification of pharmaceutical compositionssuitable for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

In one embodiment, the compositions disclosed herein may be formulatedwith a pharmaceutically acceptable carrier for administration.Pharmaceutically acceptable carriers include, but are not limited to,one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other additional ingredients that may be includedin the pharmaceutical compositions of the disclosure are generally knownin the art and may be described, for example, in Remington'sPharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, Pa.,1985, and Remington's Pharmaceutical Sciences, 20^(th) Ed., MackPublishing Co. 2000, both incorporated by reference herein.

In one embodiment, the carrier is an aqueous or hydrophilic carrier. Ina further embodiment, the carrier can be water, saline, ordimethylsulfoxide. In another embodiment, the carrier is a hydrophobiccarrier. Hydrophobic carriers include inclusion complexes, dispersions(such as micelles, microemulsions, and emulsions), and liposomes.Exemplary hydrophobic carriers include inclusion complexes, micelles,and liposomes. See, e.g., Remington's: The Science and Practice ofPharmacy 20th ed., ed. Gennaro, Lippincott: Philadelphia, Pa. 2003,incorporated by reference herein. In addition, other compounds may beincluded either in the hydrophobic carrier or the solution, e.g., tostabilize the formulation.

The composition disclosed herein may be administered by any suitableroute including oral, parenteral, transmucosal, intranasal, inhalation,or transdermal routes. Parenteral routes include intravenous,intra-arteriolar, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, intrathecal, and intracranialadministration. A suitable route of administration may be chosen topermit crossing the blood-brain barrier. Rapoport et al., J. Lipid Res.(2001) vol. 42, pp. 678-685.

The composition may be formulated according to conventional methods, andmay include any pharmaceutically acceptable additives, such asexcipients, lubricants, diluents, flavorants, colorants, buffers, anddisintegrants. See e.g., Remington's Pharmaceutical Sciences, 20^(th)Ed., Mack Publishing Co. 2000.

In some embodiments, the composition disclosed herein is formulated in asolid oral dosage form. For oral administration, the composition maytake the form of a tablet or capsule prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods generally known in the art.

In some embodiments, the composition is formulated into a liquidpreparation. Liquid preparations for oral administration may take theform of, for example, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-phydroxybenzoates or sorbic acid). The preparations may alsocomprise buffer salts, flavoring, coloring and sweetening agents asappropriate.

In other embodiments of the present disclosure, the composition may beformulated for parenteral administration such as bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The composition may take such forms as suspensions,solutions, dispersions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents.

In some embodiments, the composition may be formulated as a depotpreparation. Such formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. For example, the composition may be formulated with suitablepolymeric or hydrophobic material (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In another embodiment, the composition may be delivered in a vesicle,such as a micelle, liposome, or an artificial low-density lipoprotein(LDL) particle. See, e.g., U.S. Pat. No. 7,682,627.

In another aspect, the present disclosure is directed to a method fortreating NPC in a subject in need of treatment, comprising administeringto the subject a composition comprising bryostatin-1 in an effectiveamount to treat NPC, wherein the effective amount results in aconcentration of bryostatin-1 ranging from about 0.1 nM to about 1.5 nM.For example, in some embodiments, the effective amount results in aconcentration of bryostatin-1 in the brain of about 0.1 nM, about 0.2nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM,about 1.3 nM, about 1.4 nM, about 1.5 nM, or any value in between.

As described above, the blood plasma concentration of bryostatin-1corresponds to about five times the concentration of bryostatin-1 in thebrain. Thus, in some embodiments, an effective amount of bryostatin-1 totreat NPC results in a blood plasma concentration of about 0.5 nM toabout 7.5 nM. For example, in some embodiments, the effective amount ofbryostatin-1 results in a blood plasma concentration of bryostatin-1 ofabout 0.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, about 5 nM, about 5.5 nM,about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, or any value inbetween.

In some embodiments, the effective amount of bryostatin-1 to treat NPCranges from about 5 μg/m² to about 120 μg/m². For example, in someembodiments, the effective amount of bryostatin-1 to treat NPC is about5 μg/m², about 10 μg/m², about 15 μg/m², about 20 μg/m², about 25 μg/m²,about 30 μg/m², about 35 μg/m², about 40 μg/m², about 45 μg/m², about 50μg/m², about 55 μg/m², about 60 μg/m², about 65 μg/m², about 70 μg/m²,about 75 μg/m², about 80 μg/m², about 85 μg/m², about 90 μg/m², about 95μg/m², about 100 μg/m², about 105 μg/m², about 110 μg/m², about 115μg/m², about 120 μg/m², or any value in between.

In some embodiments, the concentrations of bryostatin-1 disclosed hereinare peak concentrations of bryostatin-1

The compositions and methods described herein will be further describedby the following examples.

EXAMPLES Example 1 Dose Kinetics and PKC Isozyme Specificity ofBryostatin-1 in Primary Human Neurons

PKC isozyme activation by bryostatin-1 at various concentrations inprimary human neurons was evaluated. One month old primary human neuronswere treated with 0 nM, 0.060 nM, 0.125 nM, 0.25 nM, 0.5 nM and 1.0 nMbryostatin 1 for 24 hr. Neurons were separated into soluble and membranefractions and immunoblotted against PKC α and PKC ε. PKC activation wasmeasured as the percentage of total protein in the membrane and reportedas percentage of control. PKC staining levels were measureddensitometrically. Data are represented as mean±SE of three independentexperiments (Students t-test, *P<0.05 and **P<0.005).

As shown in the results in FIG. 1, bryostatin-1 activated both PKC ε andPKC α. At low concentrations, bryostatin-1 showed potent PKC εactivation. At about 1 nM, bryostatin-1 exhibited potent activation ofboth PKC ε and PKC α.

Example 2 Time Kinetics and PKC Isozyme Specificity of 0.25 nMBryostatin-1 in Primary Human Neurons

PKC isozyme activation by bryostatin-1 at 0.25 nM in primary humanneurons was evaluated. One month old primary human neurons were treatedwith ethanol (C), bryostatin 1 (0.25 nM) for 1 hr, 4 hr and 24 hr.Neurons were separated into soluble (S) and membrane (P) fractions andimmunoblotted against PKC α, PKC ε and PKC δ. PKC activation wasmeasured as the percentage of total protein in the membrane and reportedas percentage of control. PKC staining levels were measureddensitometrically.

The results are shown in FIG. 2A (Western blot) and FIG. 2B (time courseof activation). Bryostatin-1 (F(3,8)=22.5; ANOVA p=0.0003) induced PKC-εactivation at 1 hr, 4 hr and 24 hr. Data are represented as mean±SE ofthree independent experiments (Students t-test, *P<0.05 and **P<0.005).

Example 3 PKC Isozyme Specificity of 0.27 nM Bryostatin-1 in SH-SY5YCells

PKC isozyme activation by bryostatin-1 at 0.27 nM in SH-SY5Y cells wasevaluated. SH-SY5Y cells are human-derived cell line and are often usedas in vitro models of neuronal function and differentiation. Cells weretreated with bryostatin-1 (0.27 nM) for 0, 5, 15, 30 and 60 min. Sampleswere fractionated into cytosol and membrane fractions and analyzed withPKC isoform-specific antibodies. Three independent experiments wereperformed for each sample.

The results are shown in FIG. 3. Bryostatin-1 activated both PKC ε andPKC α. The data in the graph represent mean±SE. (Students t-test*p<0.05; **p<0.005 and ***p<0.0005).

Example 4 PKC Isozyme Activation by Other PKC Activators

PKC isozyme activation by other PKC activators was evaluated. FIGS. 4Aand 4B show the results for different concentrations of DHA-CP6 methylester (a docosahexaenoic acid derivative) and DCPLA (a linoleic acidderivative), respectively:

DHA-CP6-ME and DCPLA were incubated with recombinant PKC isozymes (α,βII, γ, δ, and/or ε) for 5 min at 4° C. PKC activity was measuredenzymatically by measuring the incorporation of 32P-inorganic phosphatefrom 32P-ATP into histones.

FIG. 4C shows the results for different concentrations of DCPLA-ME:

For measuring PKC activation, recombinant PKC α, ε, and δ were used.DCPLA-ME induced activation was measured in absence of diacylglycerol(DAG) and phosphatidylserine (PS). Individual enzymes were incubated for15 min at 37° C. in the presence of 10 uM histones, 4.89 mM CaCl2, 10 mMMgCl2, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8ug/ml aprotinin, 8 ug/ml leupeptin, 2 mM benzamidine and 0.5 uCi of[gamma-32P]ATP.[32P]Phosphoprotein formation was measured by adsorption ontophosphocellulose. Data are represented as mean±SE of three independentexperiments (Students t-test, *P<0.05 and **P<0.005).

Example 5 PKC Activation by Bryostatin-1 in Mouse Brain

Mice were injected with bryostatin-1 in DMSO at doses of 3, 5, 10, 20,and 40 μg/m² in the tail vein. After 30 or 120 min, the brains werefrozen, then homogenized in 10 mM tris-HCl pH 7.4. The homogenates werefractionated into cytosolic and membrane fractions byultracentrifugation. Fractions were analyzed by Western blotting usingisozyme-specific antibodies. The results are shown in FIG. 5A (30 min)and 5B (120 min).

Example 6 Time Course of PKC Activation by Bryostatin-1 in Mouse Brain

Mice were injected with bryostatin-1 in DMSO at doses of 10 or 15 μg/m²in the tail vein. After 15, 30, 60, or 120 min, the brains were frozen,then homogenized in 10 mM tris-HCl pH 7.4. The homogenates werefractionated into cytosolic and membrane fractions byultracentrifugation. Fractions were analyzed by Western blotting usingPKC epsilon-specific antibodies (FIG. 6A) or PKC alpha-specificantibodies (FIG. 6B). PKC staining levels were measureddensitometrically.

Experimental Procedures

Culture of primary human cortical neurons: Human primary neurons(ScienCell Research Laboratories, Carlsbad, Calif., USA) were plated onpoly-L-lysine coated plates and were maintained in neuronal medium(ScienCell Research Laboratories, Carlsbad, Calif., USA) supplementedwith the neuronal growth supplement (NGS, ScienCell ResearchLaboratories, Carlsbad, Calif., USA). For maintenance of neurons, halfof the media was changed every 3 days. Fresh activators were added withevery media change.

Cell culture and treatments: Human SH-SY5Y neuroblastoma cells (ATCC)were cultured in 45% F12K, 45% DMEM and 10% FBS.

Cell lysis and Western blot analysis: Cells were harvested inhomogenizing buffer (HB) containing 10 mM Tris-Cl (pH 7.4), 1 mM PMSF(phenylmethylsulfonylfluoride), 1 mM EGTA, 1 mM EDTA, 50 mM NaF and 20μM leupeptin, and were lysed by sonication. The homogenate wascentrifuged at 100,000×g for 15 min at 4° C. to obtain the cytosolicfraction (supernatant) and membrane (pellet). The pellet was resuspendedin the HB by sonication. Protein concentration was measured using theCoomassie Plus (Bradford) Protein Assay kit (Pierce, Rockford, Ill.,USA). Following quantification, 20 μg of protein from each sample wassubjected to SDS-PAGE analysis in a 4-20% gradient Tris-Glycinepolyacrylamide gel (Invitrogen, Carlsbad, Calif., USA). The separatedprotein was then transferred to a nitrocellulose membrane. The membranewas blocked with BSA and incubated with primary antibody overnight at 4°C. After incubation, it was washed 3× with TBS-T (Tris-bufferedsaline-Tween 20) and further incubated with alkaline phosphataseconjugated secondary antibody at 1:10000 dilution for 45 min. Themembrane was finally washed 3× with TBS-T and developed using the 1-stepNBT-BCIP substrate (Pierce, Rockford, Ill., USA). The blot was imaged ina ImageQuant RT-ECL (GE Life Sciences, Piscataway, N.J.) anddensitometric quantification was performed using IMAL software. Forquantifying expression of a protein, the densitometric value for theprotein of interest was normalized against beta-actin (loading control).

PKC assay: For measurement of PKC activation by DCPLA-ME, activation ofrecombinant PKCalpha, PKCepsilon and PKCdelta (Cell SignalingTechnology, USA) was used. DCPLA-ME induced activation was measured inabsence of diacylglycerol (DAG) and phosphatidylserine (PS). Individualenzymes were incubated for 15 min at 37° C. in the presence of 10 uMhistones, 4.89 mM CaCl2, 10 mM MgCl2, 20 mM HEPES (pH 7.4), 0.8 mM EDTA,4 mM EGTA, 4% glycerol, 8 ug/ml aprotinin, 8 ug/ml leupeptin, 2 mMbenzamidine and 0.5 uCi of [gamma-32P]ATP. [32P]Phosphoprotein formationwas measured by adsorption onto phosphocellulose.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A composition comprising bryostatin-1 in aneffective amount to treat Niemann-Pick Type C (NPC), wherein theeffective amount results in a concentration of bryostatin-1 in the brainof a subject in need of treatment ranging from about 0.1 nM to about 1.5nM.
 2. The composition of claim 1, wherein the effective amount resultsin a concentration of bryostatin-1 in the brain of about 1 nM.
 3. Amethod for treating Niemann-Pick Type C (NPC) in a subject in need oftreatment, comprising administering to the subject a compositioncomprising bryostatin-1 in an effective amount to treat NPC, wherein theeffective amount results in a concentration of bryostatin-1 in the brainranging from about 0.1 nM to about 1.5 nM.
 4. The method of claim 3,wherein the effective amount results in a concentration of bryostatin-1in the brain of about 1 nM.